Chromosome replication and transmission are essential for the inheritance of genetic traits, but the mechanisms responsible for these processes remain poorly understood in multicellular eukaryotes. The centromere is required for kinetochore formation, which serves as the key attachment site to the spindle during mitosis and meiosis. Defects in centromere or kinetochore function result in aneuploidy, which is a hallmark of human cancers and is responsible for many birth defects. A pressing question in the centromere field today is how centromere identity is propagated from one generation to the next in multicellular eukaryotes. Our published and unpublished results demonstrate that the levels of centromeric chromatin proteins and their regulators are tightly regulated by gene expression and proteolytic mechanisms to ensure faithful centromere and chromosome propagation. This proposal integrates genetic, molecular, cell biological and biochemical approaches to identify the molecules and mechanisms that regulate the levels of centromeric chromatin proteins, and promote the assembly and propagation of centromeric chromatin, using Drosophila cell culture and animal tissues. The entry point into centromeric chromatin is a conserved histone H3-like protein (CENP-A, CID in flies) that localizes exclusively to functional centromeres. We previously identified key regulators of CENP-localization and assembly, as well as a link between cell cycle regulation and centromere formation, which provide an intellectual and technical foundation for the proposed aims. The specific focus of this proposal is to elucidate the cell cycle regulation of centromeric protein stability and the consequences of misregulation by investigating: 1) how mutual protection and other mechanisms regulate the stability of CID and its chaperone CAL1, 2) the impact of centromere protein misregulation on cells, tissues and the organism, and 3) how centromere protein misregulation contributes to cancer initiation and progression in Drosophila model of glioblastoma. The deeper understanding of the normal regulation of centromere assembly generated by these studies will provide basic information about this essential biological process, insights into mechanisms responsible for the etiology of aneuploidy associated with cancer and birth defects, and ultimately will lead to the development of tools for diagnosis and treatment of human diseases associated with centromere defects, such as cancer.